Some of the projects carried over and continued from the previous year but new collaborations were also initiated with several intramural researchers during this year. Our NIDCR (Yamada Lab) collaboration on cell durotaxis (cell motile response to substrate stiffness gradients) required revisiting some of the experiments conducted before a paper was submitted. Questions were raised as to the role of the mean substrate stiffness in addition to the that of stiffness gradients. Several new gradient gels with different mean elastic moduli were prepared and we used the AFM to map their elasticity before cell motile response to those substrates was investigated. It was found that, for each cell line, there was an upper limit of mean stiffness beyond which the cells remained relatively indifferent to the local gradients. A paper detailing the results of the study was accepted and published by the Biophysical journal during 2019. Our collaboration with NCI (Dalal Lab) on the various aspects of the structure/stoichiometry of the centromere in the past year focused on the mechanics of the centromere vs the canonical nucleosome. Extramural collaborators of the NCI lab used computational methods to show that the two complexes were thermodynamically different resulting in the centromere being more rigid. We used the AFM to probe whether we can detect a difference in apparent elasticity of reconstituted nucleosomes with incorporated recombinant H3 or cenP-A histones. We managed to obtain high-resolution elasticity profiles across single nucleosomes and the differences were apparent and consistent with the in-silico results. A paper including our AFM force spectroscopy results is under revision. We initiated a new project with NIDDK (Hinton Lab) to study interactions of certain bacteriophage proteins with DNA and with certain bacteria proteins. There has been renewed interest in and some recent successes with the old idea of using bacteriophages as therapeutic agents against antibiotic-resistant bacteria. Previous work by the Hinton lab studied the early expressed T4 phage protein MotB and found that heterologous MotB expression is highly toxic to E-coli and that it binds to both host and phage (modified - glucosylated, hydroxymethylated-5 cytosine or GHme-C) DNA. They also found that a histone-like bacterial protein H-NS copurifies with MotB leading to the hypothesis that MotB may be interfering with the critical functions of H-NS. We undertook an extensive imaging project to visualize the various interactions among the three macromolecules (DNA, H-NS and MotB). We examined modes of interactions with both unmodified and modified DNA. The reaction with H-NS gave the expected pattern of distinct bridging of DNA chains, while MotB bound strongly to both types of DNA in a clearly cooperative manner, forming large multi-DNA complexes. The interactions were significantly enhanced when both H-NS and MotB were reacted with DNA. The reaction formed very large DNA-protein complexes that were about 2 orders of magnitude larger than when MotB acted alone. This lends strong support to the hypothesis of MotB interfering with the functions of H-NS. We are in the process of further quantifying the reaction products under different conditions and a paper is in preparation. The plan is to examine additional macromolecules playing a role in the bacteriophage-bacteria battle. We continued our collaboration with NEI (Sergeev lab) examining self-association of human Typr1 protein and possible protein-DNA interactions. Tyrp1 (tyrosinase-related protein 1) is a melanocyte enzyme involved in melanin synthesis with mutations linked to certain types of albinism. It acts in conjunction with another enzyme, tyrosinase, and little is understood about their function and possible interactions. We had previously shown, using the AFM, that tyrosinase exists as a monomer, consistent with other methods, such as analytical ultracentrifugation (AUC). During this past year we worked on Tyrp1 and showed that it also exists as a monomer. It was observed that Tyrp1 incubated at 37 degrees C forms large structures that we showed to be aggregates with a wide range of stoichiometries. We are continuing to examine possible interactions between Tyrp1 and DNA and between the two enzymes. A paper including our tyrosinase results was published during the year and another on Tyrp1 is in preparation. A new collaboration with NHLBI (Adelstein lab) focuses on the mechanics of T-cells. T-cells interact with their environment and modulate their mechanics as needed for the performance of certain tasks. In particular, we are interested in the cell elasticity modulation due to interaction with the adhesion, transmembrane glycoprotein ICAM-1, a member of the immunoglobulin family. ICAM-1 in involved in inflammatory processes and in the activation of T-cells and their transendothelial migration. The relation between ICAM-1 induced signaling and changes in the T-cell cytoskeleton that facilitate T-cell migration through endothelial barriers are not well understood. We undertook to quantify elasticity of T-cells on substrates coated with ICAM-1, using force spectroscopy with our AFM instruments. Moreover, we investigated the effect of microtubule stabilizing factors (taxol) and microtubule polymerization inhibitors (nocodazole) on T-cells bound to ICAM-1 coated surfaces. We showed that the presence of ICAM-1 induces significantly softer (2x) T-cells compared to non-specific substrates, such as poly-L-lysine. Incubation with taxol induced slightly stiffer T-cells, but the effect of nocodazole was dramatic as cell elastic modulus decreased by at least an order of magnitude. We are currently looking at dose-dependence of the observed effects. The more challenging task undertaken was the measurement of the elastic moduli of T-cells on soft substrates, in the presence and absence of ICAM-1 and other factors such as taxol. These conditions are closer to physiological and are directly relevant to in-vivo functionality.